Gynogenetic Based Screen

This type of screening provides the space and time-saving aspects of hap-loid screening, but has the added advantages of allowing the observation of the diploid phenotype combined with viable resultant embryos. Gynogenetic dip-loid embryos can be produced by subjecting eggs fertilized with UV-inacti-vated sperm to either heat-shock or high hydrostatic pressure. Heat-shock administered 13 min after fertilization suppresses the first mitotic division and, therefore, induces gynogenesis. However, a large percentage of heat-shocked embryos exhibit atypical development possibly owing to structural damage to the fertilized eggs, limiting its effectiveness as a methodology for random screening. High hydrostatic pressure delivered just after fertilization restricts the second meiotic anaphase. A significant number of early pressure- (EP) induced gynogenetic embryos also develop abnormally owing to damage to the eggs, but this is seen at a lower rate than in heat-shock treated embryos. Since crossingover occurs at the first meiotic division, only mutations that occur close to the centromere will exhibit a high rate of homozygosity in the gynogenetic embryos. This somewhat hampers the use of this technique, since the percentage of mutant embryos is directly proportional to the distance that a mutation maps from the centromere and, therefore, is unpredictable. It has consequently been used as a measure of gene distance from the centromere of known mutations. This technique also has the drawback of requiring the use of controlled pressure equipment. Production of Gynogenetic Embryos via Heat-Shock

1. Collect and UV-inactivate sperm as described in Subheading 3.2.1.

2. Collect eggs and fertilize in vitro as described in Subheading 3.2.1. Keep fertilized eggs at 28°C.

3. Approximately 10 min after fertilization, transfer the developing eggs to a 42°C water bath for 2 min.

4. Return embryos to 28°C water, and allow to develop normally. Production of Gynogenetic Embryos via Early Pressure

This requires a hydraulic press or French press. These are not easily located but perhaps one place to look is amongst the discarded equipment of a microbiology department.

Collect and UV-inactivate sperm as described in Subheading 3.2.1. Collect eggs and fertilize in vitro as in Subheading 3.2.1.

3. Place fertilized eggs in embryo water in pressure vials within pressure cylinder.

4. Apply pressure to fertilised embryo 1.5 min after fertilization to 8000 lbs./sq. in. for 4.5 min.

5. Remove embryos from vials, distribute into dishes, and allow to develop normally. With both gynogenetic diploid and haploid screening, we find it useful to separate embryos with mechanically induced defects from normal developing embryos as soon as possible. All embryos are kept and screened, but are also ranked by quality. Any consistent defect that occurs in a percentage of the embryos predicted by the screening rationale is considered mutant and rescreened. Researchers learn defects that are induced by manipulating the embryos and those that reflect a true genetic lesion.

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